Partial bladder outlet obstruction (PBOO) is an important urological complication that results from a number of pathological conditions. Stretch and hypoxia have been proposed as early initiation signals that lead to PBOO-induced bladder smooth muscle (SM) hypertrophy, although the exact mechanism is unknown. Also, we have determined in the previous grant that the expression of a number of contractile and regulatory proteins, including SM myosin, nonmuscle myosin, Rho-kinase, tropomysoin and telokin is effected by PBOO. Determining the mechanisms by which expression of these proteins are altered, what, if any, specific role they play in bladder dysfunction and whether there is a coordinated regulation of contractile protein expression in response to PBOO is the long-term goal of this project. Using a stable SM cell line that we developed from bladder (termed BSM) that retains a SM phenotype and the ability to contract and relax in response to physiological agonist, we will (1) determine if stretch and/or hypoxia, isolated from other biological events which occur during PBOO, can induce any of the molecular changes in contractile protein and/or regulatory protein expression that we have already shown to occur in vivo in response to PBOO, (2) determine if the endothelin/Rho-kinase pathway plays a role in the molecular mechanisms of stretch and hypoxia, (3) examine whether changing the amount and/or type of collagen that isolated bladder myocytes are cultured on can induce changes in the contractile and/or regulatory protein expression pattern of the BSM cells, (4) examine the effects of altered expression of smooth muscle contractile, regulatory and cytoskeletal proteins on coordinated expression and mechanical properties of the bladder smooth muscle cell line. For each of the above aims we will examine the expression of contractile and regulatory proteins at the mRNA and protein level using RT-PCR, Western blotting and Immunofluorescence microscopy. In addition, maximum isometric tension, shortening velocity, stiffness, shortening capacity, and intracellular calcium will be measured in single or bundles, of intact and demembranated cultured bladder SM cells. The results gained from this proposal will help elucidate the function, order and coordination of the molecular pathways involved in PBOO-induced bladder dysfunction.